Amino acids, phosphorylation

Posttranslational modification Examples of posttranslational modification POSTTRANSLATIONAL MODIFICATION OF POLYPEPTIDE CHAINS (p. 440) Many polypeptide chains are covalently modified after translation. Such modifications include trimming excess amino acids, phosphorylation which may activate or inactivate the protein, glycosylation which targets a protein to become part of a plasma membrane or lysosome or be secreted from the cell, or hydroxylation such as that seen in collagen. 

Proteins which act in the nucleus require specific sequences, known as nuclear localization sequences, to direct their transport from the cytoplasm to the nucleus. The nuclear localization sequences are generally found at the C-terminus of a protein and often comprise basic amino acids. Phosphorylation in sequences that are required for import into or export from the nucleus can decide whether the transcriptional activator is located predominantly in the cytoplasm or in the nucleus where it can exert its activating function. 

The procedure described is essentially that of Shioiri and Yamada. Diphenyl phosphorazidate is a useful and versatile reagent in organic synthesis. It has been used for racemlzatlon-free peptide syntheses, thiol ester synthesis, a modified Curtius reaction, an esterification of a-substituted carboxylic acld, formation of diketoplperazines, alkyl azide synthesis, phosphorylation of alcohols and amines,and polymerization of amino acids and peptides. - Furthermore, diphenyl phosphorazidate acts as a nitrene source and as a 1,3-dipole.An example in the ring contraction of cyclic ketones to form cycloalkanecarboxylic acids is presented in the next procedure, this volume. 

The phosducin polypeptide chmn, of some 240 amino acids, is folded into two domains (Figure 13.16). The N-terminal domain is mostly a-helical and appears to be quite flexible since only a weak electron density is obtained in the structure determination. The actual path of the polypeptide chain from the end of helix to the beginning of helix Ba is tentative due to slight disorder. This region is close to serine 73 at the beginning of Ba, which also becomes disordered on phosphorylation. 

FIGURE 15.2 Enzymes regulated by covalent modification are called interconvertible enzymes. The enzymes protein kinase and protein phosphatase, in the example shown here) catalyzing the conversion of the interconvertible enzyme between its two forms are called converter enzymes. In this example, the free enzyme form is catalytically active, whereas the phosphoryl-enzyme form represents an inactive state. The —OH on the interconvertible enzyme represents an —OH group on a specific amino acid side chain in the protein (for example, a particular Ser residue) capable of accepting the phosphoryl group. 

Pyruvate kinase possesses allosteric sites for numerous effectors. It is activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl-CoA, and alanine. (Note that alanine is the a-amino acid counterpart of the a-keto acid, pyruvate.) Furthermore, liver pyruvate kinase is regulated by covalent modification. Flormones such as glucagon activate a cAMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the enzyme. The phos-phorylated form of pyruvate kinase is more strongly inhibited by ATP and alanine and has a higher for PEP, so that, in the presence of physiological levels of PEP, the enzyme is inactive. Then PEP is used as a substrate for glucose synthesis in the pathway (to be described in Chapter 23), instead... 

Immunreceptor based activation motif. The classical ITAM motif comprises the consensus sequence Yxxl/ Lx(6-12)YxxI/L (where Y stands for tyrosine, I stands for isoleucine, L stands for leucine, and x can be any amino acid). Kinases containing tandem SH2 domains, as for example ZAJP-70 or SYK, recognize the phosphorylated ITAMs, thereby initiating downstream signaling events. 

In addition, eNOS is subject to protein phosphorylation. It can be phosphotylated on several serine (Ser), threonine (Thr), and tyrosine (Tyr) residues however, major changes in enzyme function have been reported for the phosphorylation of amino acid residues Seri 177 and Thr495 (in the human eNOS sequence) (Fig. 3). 

Pleckstrin homology domain (PH-domain) was first identified at the amino and carboxyl termini of a haematopoietic protein called pleckstrin. PH-domain, a protein region of approximately 120 amino acids, by binding to phosphatidylinositol lipids of the biological membranes induces the translocation of the PH-domain containing protein to membrane compartment. Various PH-domains possess specificities for phosphoinositides phosphorylated at different sites within the inositol ring. 

---